Lab

PYR PROCEDURE

Effective Date: 
Wed, 04/15/2020
Policy: 

Purpose and Scope:

PYR is a rapid colorimetic method for presumptive identification of enterococci and Group A Streptococcus based on the activity of the enzyme pyrolidonyl arylamidase. L-pyroglutamic acid beta-napthylamide is impregnated into the test disk and serves as the substrate for the detection of pyrolidonyl arylamidase.  Hydrolis of the substrate yields beta-naphthylamide which combines with the PYR Reagent (p-dimethylamino-cinnamaldehyde) to form a bright pink to cherry red color.  A positive PYR test allows for the presumptive identification of group A streptococci and Enterococcus spp..

Regents and Supplies:

PYR Test Kit from Hardy Diagnostics

  • PYR test disks
  • PYR Chromogenic Solution

Distilled Water

Sterile inoculating loop or wooden stick

Forceps for handling the disks

Reagent Storage:

Upon receipt, store at 2-8o.  The expiriation date applies to the product in its intact packaging when stored as directed.  The product may be used and tested up to the expiration date on the label.

Precautions:

Do not ingest, inhale, or allow to come into contact with skin. Handle observing the usual universal blood precautions.

 

Procedure: 

Specimen Collection:

This product is not intended for primary isolation of patient specimens.  It should be used only with cultures of isolated organiss.  This product is used in conjuction with other biochemical tests to identify cultures of isolated organisms.

Method of Use:

  1. Moisten the PYR Disk slightly with distilled or deionized water. Do not saturate.
  2. Pick 2-3 well isolated, 18-24 hour colonies, grown on Blood Agar.  Rub into a small area of the PYR disk so that there is a visible paste.
  3. Allow to react for two minutes.
  4. Add on drop of Chromogenic Solution (PYR Reagent)

Interpretation of Results:

A bright pink or cherry red color will appear within one minute if the test is positive.  A negative test is indicated by no color change.  The development of an orange, salmon, or yellow color should be interpreted as a negative reaction.

Organisms expected to give a positive result:

  • Group A streptococci (Streptococcus pyogenes
  • Enterococcus spp.

Quality Control:

Negative Control:  Group B Strep (Streptococcus agalatiae ATCC 12386)

Positive Control:  Enterococcus faecalis ATCC 29212

QC should be performed each day of use and recorded in the QC binder.

Key Points: 

Limitations:

  • PYR may be used in the presemptive separation of group A streptococci and Enterococcus spp. from other streptococci.  Additional testing, using a pure culture, is recommended for complete identification.
  • A false negative test can result if the disk is too moist.
  • False negative tests can result if selective media is used to provide inocula.

References:

Kit Insert, Instructions for Use, PYR Test Kit and PYR Reagent, Hardy Diagnostics, 1996  https://catalog.hardydiagnostic.com/cp_prod/content/hugo/PYRTestKit_Rgnt.htm

 

STREPPRO LANCEFIELD GROUP A TEST

Effective Date: 
Wed, 03/04/2020
Policy: 

Hardy Diagnostics StrepPRO Grouping Kit provides a rapid latex agglutination method for the serological identification of Lancefield group A from isolated colonies of beta-hemolytic Stretococcus species.

Beta-hemolytic streptocicci are the most frequently isolated human pathogens among the representatives of the genus Streptococcus.  Nearly all the beta-hemolytic streptococci possess specific carbohydrate antigens, known as streptococcal grouping antigens.

Hardy Diagnostics StrepPRO Grouping Kit liberates the specific antigen from the bacterial cell wall using a modified nitrous acid extraction.  The extracted antigen, in conjunction with latex agglutination, offers a rapid, sensitive, and specific method for identification of streptococcal group A from primary culture plates.  Extraction Reagents 1 and 2 contain a chemical substance able to extract the streptococcal group specific antigen at room temperature.  Extraction Reagent 3 contains a neutralizing solution.  The neutralized extract can then be identified using blue latex particles sensitized with purified group specific rabbit immunoglobulins.  These blue latex particles agglutinate strongly in the presence of homologous antigen and will not agglutinate when homologous antigen is absent.

MATERIAL SUPPLIED:

  • Blue Latex Suspension Group A
  • Extraction Reagent 1
  • Extraction Reagent 2
  • Extraction Reagent 3
  • Polyvalent Positive Control
  • Latex test Card

MATERIALS REQUIRED BUT NOT SUPPLIED

  • Standard microbiological culture supplies
  • 12X75mm test tubes
  • Timers
  • Applicator sticks

STORAGE AND SHELF LIFE

Upon receipt, store at 2-80 C away from direct light.  Do Not Freeze.  This kit, or any of its reagents, should not be used if there are any signs of discoloration, contamination, or if the expiration date has passed.

When stored as directed, the product may be used and tested up to the expiration date on the product label.

PRECAUTIONS

The Blue Latex Reagents, Extraction Reagent 1, and Extraction Reagent 3 contain sodium azide as a preservative.  Sodium axide can react explosively with copper or lead if allowed to accumulate.  Although the amount of sodium azide in the reagents is minimal, large quatities of water should be used when flushing these reagents down the sink.

Extraction Reagents Reagents 1, 2, and e contain a caustic agent.  In case of skin contact, immediately wash with soap and copious amounts fo water.  In case of eye contact, flush for at least 15 minutes with water.

 

 

 

 

 

Procedure: 

In general, an overnight, gram-positive, beta-hemolytic isolate of streptococcal isolate is required for use in this assay.  Colonies should be chosen from an area demonstrating obvious isolation.  One to four isolated colonies are recomended for grouping testing; however, if colonies are pinpoint, an increased number of colonies, approximately a loopful, should be used.

TEST PROCOTOL:

  1. Allow all reagents to acclimatize to room temperature for 10 minutes prior to use.
  2. Label one 12X75mm test tube for each specimen tested.
  3. Add one drop of Extraction Reagent 1 to each tube.
  4. Using a loop, select 1 to 4 isolated beta-hemolytic colonies and suspend them in Extraction Reagent 1.  If colonies are minute, pick a loopful of colonies, such that the Extraction Reagent 1 becomes turbid.
  5. Add one drop of Extraction Reagent 2 to each tube.
  6. Mix the reaction by tapping the tube with a finger for 10 seconds.
  7. Add five drops of Extraction 3 to each tube.  Mix the reaction as in step 6.
  8. Prior to use, resuspend the Blue Latex Reagent A by inverting the tubes.  Dispense one drop of the latex suspesion onto separate circles on the test card.
  9. Using a pasteur pipette, place one drop of the extract suspension next to the drop of latex suspension in each circle. Ensure that the pipette tip does not touch the latex suspension.
  10. Mix the blue latex and the extract with a wooden applicator stick, using the complete area of the circle.
  11. Gently hand rock the entire card allowing the mixture to flow slowly within the ring area.
  12. At one minute, under normal lighting condidtions, observe for agglutination, or strong clumping, of the blue latex particles.

INTERPRETATION OF RESULTS

Positive Results:

A rapid and significantly stong clumping of the blue latex particles, to form an agglutination pattern indicates an identification of the streptococcal isolate for Lancefield group A.

A weak reaction should be repeated using a heavier inoculum.  The repeated test is considered positive if a visible agglutionation occurs.

Negative Results:

No visible agglutination of the latex particles indicates a negative reaction for Lancefield group A.

Key Points: 

QUALITY CONTROL

Test the kit each day of use with a known positive - a colony from the quality control Group A Strep plate, and with a known negative - a colony from the quality control Group B Strep plate.  Record in the StrepPRO quality control log.

LIMITATIONS

The kit is intended for use in the identification of beta-hemolytic streptococci from a Blood Agar Plate.

CLIA CLASSIFICATION FOR IN-HOUSE TESTING

Effective Date: 
Thu, 03/01/2018
Reviewed: 
Thu, 03/01/2018

ASYMPTOMATIC SELF-DIRECTED STI TESTING

Effective Date: 
Thu, 09/21/2017
Revised: 
Tue, 12/04/2018
Policy: 

This Student Health Center policy allows patients to order their own testing for 4 sexually transmitted infections: HIV, Syphilis, Chlamydia and Gonorrhea.  This policy includes permission for patients to order and to self-collect rectal and/or vaginal swabs for Gonorrhea and/or Chlamydia testing.  The policy reviews the RNs role in reviewing self-directed STI results.

 

Procedure: 

1. The Student Health Center web page educates students about what tests are available, when to be tested, and what body parts to test. 

2. SHOP provides further support to students with additional questions or to those who need assistance in identifying which tests are indicated.

3. Student completes the online ordering questionnaire via their Health e-Messenger account and presents at the lab.

4. Lab provides appropriate supplies and instructions for proper self-collection of urine, vaginal or rectal specimens.

5. Lab draws blood for HIV and/or syphillis testing.

6. Patients needing a throat swab for gonorrhea are directed to the triage nurse, who will collect the sample under their Standardized Procedures for Throat Culture for Strep and Gonorrhea.

7. Lab processes samples under standing order from the Medical Director.

8. RNs review Self-Directed STI lab results every morning. See Standardized Procedure: Nurse Ordering and Reviewing Labs and X-ray.

9. Patients with negative test results receive appropriate, standardized secure message with results, using the Health e-Messenger system.  Positive test results are forwarded to the Medical Director or Patient Care Coordinator who contacts the student for appropriate treatment.

Some patients are not appropriate for self-directed STI testing.  The following patients should be referred for a clinician visit:

  • Patients with symptoms
  • Patients with complex issues
  • Patients who are presenting as a known contact of someone with an STI
  • Patients who request hepatitis or herpes testing
  • Patients who are victims of sexual assault

Key Points:

CDC Guidelines recommend that all women under age 26 be tested annually for chlamydia and gonorrhea.

Men who have sex with men are recommended to test every 3 to 4 months.

Rapid diagnosis and treatment of STIs is a public health imperative. Barriers to testing, such as requirement for a clinician visit, remain a significant source of delayed identification and treatment of STIs, particularly HIV. The implementation of self-directed at the  UCSC Student Health Center expedites timely STI Testing.

QUANTITATIVE D-DIMER ASSAY **

Effective Date: 
Fri, 03/24/2017
Reviewed: 
Wed, 02/05/2020
Revised: 
Fri, 01/12/2018
Policy: 

ALERE TRIAGE® D-DIMER TEST

The Alere Triage® D-Dimer Test is a fluorescence immunoassay to be used with the Alere Triage® Meters for the quantitative determination of cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood and plasma specimens. The test is used as an aid in the assessment and evaluation of patients suspected of having disseminated intravascular coagulation or thromboembolic events including pulmonary embolism

Procedure: 

 

TEST PRINCIPLE

The test procedure involves the addition of several drops of an EDTA anticoagulated whole blood or plasma specimen to the sample port on the Test Device. After addition of the specimen, the whole blood cells are separated from the plasma using a filter contained in the Test Device. The specimen reacts with fluorescent antibody conjugates and flows through the Test Device by capillary action. Complexes of fluorescent antibody conjugate are captured on a discrete zone specific to that analyte.

REAGENT

The Alere Triage® D-Dimer Test Device contains all the reagents necessary for the quantification of cross-linked fibrin degradation products containing D-dimer in EDTA anticoagulated whole blood or plasma specimens

INSTRUMENT OPERATION

The Test Device is inserted into the Alere Triage® Meter (hereafter referred to as Meter). The Meter is programmed to perform the analysis after the specimen has reacted with the reagents within the Test Device. The analysis is based on the amount of fluorescence the Meter detects within a measurement zone on the Test Device. The concentration of the analyte in the specimen is directly proportional to the fluorescence detected. The results are displayed on the Meter screen in approximately 20 minutes from the addition of specimen. All results are stored in the Meter memory to display or print when needed. If connected, the Meter can transmit results to the lab or hospital information system.

SAMPLE COLLECTION AND HANDLING

SAMPLE TYPE

A venous whole blood or plasma specimen using K2 EDTA as the anticoagulant is acceptable for testing with this product. Other blood specimen types have not been evaluated.

COLLECTION

For specimen collection, follow the sample tube manufacturer’s recommended procedure.

STORAGE AND STABILITY

If using whole blood, test patient specimen within 24 hours of sample collection. If testing cannot be completed within 24 hours, the plasma should be separated and stored at -20°C until it can be tested. No more than a single freeze/thaw cycle is recommended.

After testing is complete, hold whole blood same for 24 hours for possible retest.  If plasma is used and has not been frozen prior to testing, freeze plasma sample and hold for 30 days.

HANDLING PRECAUTIONS

Transport specimens at room temperature or chilled and avoid extreme temperatures.

Avoid using severely hemolyzed specimens whenever possible. If a specimen appears to be severely hemolyzed, another specimen should be obtained and tested.

REAGENTS AND EQUIPMENT

REAGENTS AND MATERIALS PROVIDED

Alere Triage® D-Dimer Test Device kit

Quantity: 25 Test Devices

Description of reagent: Single Use test device

Murine monoclonal antibodies against D-Dimer

Fluorescent dye

Stabilizers

Storage requirements:

Store the Test Devices in a refrigerator at 2-8°C (35-46°F).

Once removed from refrigeration, the pouched Test Device is stable for up to 14 days at room temperature, but not beyond the expiration date printed on the pouch. With a soft, felt tip marker, gently write the date and time of removal from the refrigerator on the pouch and cross out the manufacturer expiration date printed on the pouch. Care must be taken to document the time the product is at room temperature. Once equilibrated to room temperature, do not return the Test Device to refrigeration.

Before using refrigerated Test Devices, allow individual foil pouches to reach operating temperature (20-24°C or 68-75°F). This will take a minimum of 15 minutes. If a kit containing multiple Test Devices is removed from refrigeration, allow the kit to reach room temperature before use. This will take a minimum of 60 minutes.

Do not remove the Test Device from the pouch until prepared for immediate use.

Discard after single use.

Special handling requirements:

For In Vitro Diagnostic Use.

For use by healthcare professionals.

Do not use the kit beyond the expiration date printed on the outside of the box.

Carefully follow the instructions and procedures described in this document and the package insert.

Optimal results will be achieved by performing testing at temperatures between 20-24ºC (68-75 ºF).

If results from multiple samples within the same patient will be compared, it is recommended to maintain a consistent sample type (whole blood or plasma).

Sample dilution is not recommended.

Transfer pipettes

Quantity: 25

Storage requirements: room temperature

Special handling requirements:

The transfer pipette should be used for one patient specimen only. Discard after single use.

Reagent CODE CHIP™ Module

Quantity: 1

Paper Roll

Quantity: 1

QUALITY CONTROL

ALERE TRIAGE® QC DEVICE

Use the QC Device to ensure proper function of the Meter. Perform QC Device testing for the following conditions:

Upon initial setup of the Meter.

Each day of patient testing.

When the Meter has been transported or moved.

Whenever there is uncertainty about the performance of the Meter.

Whenever required by your laboratory’s quality control requirements.

Do not discard the Alere Triage® QC Device and associated CODE CHIP™ module. Store them in the QC Device Box.

Refer to the Alere Triage® Meter User Manual for complete instructions for use of the QC Device.

The first time a new QC Device is run in the Meter, install the QC Device CODE CHIP™ module. The QC Device CODE CHIP™ module data is stored in the Meter memory. The QC Device CODE CHIP™ module does not need to be reinstalled after the first time.

Installing QC Device CODE CHIP™

From the main screen, select Install New Code Chip and press Enter.

Place the QC Device CODE CHIP™ module into the lower left front corner of the Meter. Follow the prompts on the screen.

Remove the QC Device CODE CHIP™ module from the Meter when data transfer is complete.

Place the QC Device CODE CHIP™ module back into the QC Device Box for storage.

From the main screen, select Run Test and press Enter.

If User ID is enabled enter your User ID number and press Enter.

Select QC Device and press Enter.

Insert QC Device into the Meter and press Enter.

A Pass or Fail result will be displayed when complete. Each parameter should pass before patient testing is performed.

Remove the QC Device from the Meter and place in the QC Device Box. DO NOT DISCARD THE QC DEVICE.

Note: If the QC Device or external controls do not perform as expected, review the above instructions to see if the test was performed correctly, repeat the test, then contact Alere or your local Alere representative (refer to Contact Alere section). Refer to the Alere Triage® Meter User Manual for a complete description of the quality control system.

INTERNAL PROCEDURAL CONTROLS

Every Alere Triage® D-Dimer Test is a quantitative test that includes two control materials of different concentrations that are run automatically with every patient specimen, external liquid control solution, or proficiency testing sample. If the automatic check of these built-in controls shows that the control value results are within the limits set during manufacturing, the Meter will report a result for the specimen or sample being tested. If the automatic check of these built-in controls shows that the control value results are not within the limits set during manufacturing, a test result will not be reported. Instead, the Meter will display a warning or error message that is described in the Alere Triage® Meter User Manual.

Users should follow government guidelines (for example, federal, state or local) and/or accreditation requirements for quality control.

EXTERNAL LIQUID CONTROLS

Good Laboratory Practice suggests that external controls should be tested with each new lot of test materials, or every 30 days, and as otherwise required by your laboratory’s standard quality control procedures. Controls should be tested in the same manner as if testing patient samples. When running patient specimens or external controls, if an analyte fails for any reason (built-in control failure or an external control out of range) no patient results will be reported.

The use of non-Alere Controls and Calibration Verification materials is not recommended.

CALIBRATION

Lot Calibration Using the Reagent CODE CHIP™ Module

When a new lot of Test Devices is opened, the calibration and expiration data for that lot of Test Devices must be transferred to the Meter before patient testing. Use the Reagent CODE CHIP™ module supplied with the new lot of Test Devices to transfer the data to the Meter.

Installing the Reagent CODE CHIP™

Perform one time for each new lot of Test Devices

From the main screen, select Install New Code Chip. Press Enter.

Place the Reagent CODE CHIP™ module into the lower left front corner of the Meter and follow the prompts on the screen.

Remove the Reagent CODE CHIP™ module from the Meter when data transfer is complete.

Place the Reagent CODE CHIP™ module back into its original container for storage.

PRECAUTIONS

For professional in vitro diagnostic use only.

Patient specimens, used Test Devices and used transfer pipettes are potentially infectious. Proper handling and disposal methods should be followed in accordance with local, state and federal regulations.

Proper laboratory safety techniques should be followed at all times when working with patient specimens because they are potentially infectious.

The Alere Triage® D-Dimer Test should not be used as absolute evidence for PE or DVT. As with all in vitro diagnostic tests, the test results should be interpreted by the physician in conjunction with clinical findings and other test results.

PERFORMING A TEST

TESTING INSTRUCTIONS

Procedural Notes

For each day of patient testing, perform QC Device testing. Refer to the Quality Control section.

Frozen plasma and refrigerated whole blood or plasma specimens must be allowed to reach room temperature and be mixed thoroughly before testing.

Mix whole blood specimens by gently inverting the tube several times before testing.

It is recommended to mix plasma specimens by vortexing the tube before testing.

Add Patient Specimen

Open the pouch and label the Test Device with the patient identification number.

Place the Test Device on a level, horizontal surface.

Using the transfer pipette, squeeze the larger (top) bulb completely and insert the tip into the specimen.

Release the bulb slowly. The transfer pipette barrel should fill completely with some fluid flowing into the smaller (lower) bulb.

Place the tip of the transfer pipette into the sample port of the Test Device and squeeze the larger bulb completely. The entire volume of fluid in the transfer pipette barrel must flow into the sample port. The specimen in the smaller (lower) bulb will not be expelled.

Remove the transfer pipette tip from the sample port and then release the larger (top) bulb.

Discard the transfer pipette.

Allow specimen to absorb completely before moving the Test Device.

Run Test

From the main screen, select Run Test and press Enter.

If User ID is enabled enter your User ID number and press Enter.

Select Patient Sample and press Enter.

Enter the patient identification number and press Enter.

Confirm that the number was entered correctly by selecting Confirm Patient ID and pressing Enter. If the number was not entered correctly, select Correct Patient ID, press Enter and repeat the previous step.

Holding the Test Device by the edges, insert the Test Device into the Meter and press Enter. The results will be displayed when the analysis is complete.

Note: The Test Device must be inserted into the Meter within 30 minutes from the time the patient specimen was added. A delay longer than 30 minutes may cause the results to be invalid and blocked out on the printout.

Read The Results

Repeat Critical Results

Results may be printed by pressing the Print button.

Discard the Test Device after release from the Meter.

A blocked out result indicates the result was invalid and the test should be repeated.

RESULTS

EXPECTED VALUES

Normal: </=500ng.mL DDU

A normal D-Dimer result has a negative predictive value of approximately 95% for the exclusion of acute pulmonary embolism (PE) or deep vein thrombosis when there is low or moderate pretest PE probability.

The Alere Triage® D-Dimer Test has been standardized using a purified protein preparation of D-dimer based on the mass (concentration) of the analyte present in EDTA anticoagulated plasma.

The D-dimer values are presented in units of mass (ng/mL) of D-dimer, also known as D-dimer Units (D-DU). There are no international standards for D-dimer and different assays use antibodies with differing specificities for D-dimer and other fibrin degradation products. This can lead to poor correlation between methods reporting results in D-DU.

METHOD PERFORMANCE SPECIFICATIONS

MEASURABLE RANGE

The D-Dimer range reported by the test system is 100 ng/mL to 5000 ng/mL. Due to calibration constraints, our lab will, as of 8/14/2017, report values of <171 ng/mL simply as <171 ng/mL rather than as a discrete numberical value.  This change will have no significant effects on any patient outcomes with values </=500 ng/mL  reported before this time.)

LIMITATIONS

The results should be evaluated in the context of all the clinical and laboratory data available. In those instances where test results do not agree with the clinical evaluation, additional tests should be performed.

Severely hemolyzed specimens should be avoided. When a sample appears to be severely hemolyzed, another specimen should be obtained and tested.

This test has been evaluated with venous whole blood and plasma using K2 EDTA as the anticoagulant. Other specimen types, draw methods, or anticoagulants have not been evaluated. Use standard veni-puncture techniques. Follow the sample collection recommendations of the sample tube manufacturer.

There is the possibility that factors such as technical or procedural errors, as well as substances in blood specimens that may interfere with the test and cause erroneous results. Please refer to the package insert for a list of interfering substances.

As with any assay employing mouse antibodies, the possibility exists for interference by human anti-mouse antibodies (HAMA) in the sample. Similarly, specimens from patients who have been routinely exposed to animals or to animal serum products may contain heterophile antibodies which may cause erroneous results.

This assay is a fluorescence immunoassay, and may be affected by environmental conditions.

REFERENCES

Fedullo, P.F. and V.F. Tapson. The evaluation of suspected pulmonary embolism.New England Journal of Medicine 349: 1247-1256, 2003.

S.Z. Goldhaber. Pulmonary embolism. New England Journal of Medicine 339: 93-104, 1998.

Kline, J.A., Mitchell, A.M., Kabrhel, C., Richman, P.B., and D.M. Courtney. Clinical criteria to prevent unnecessary diagnostic testing in emergency department patients with suspected pulmonary embolism. Journal of Thrombosis and Haemostasis 2(8):1247-1255, 2004.

Ramzi, D.W. and K.V. Leeper. DVT and pulmonary embolism: Part I. Diagnosis. American Family Physician 69(12): 2829-2836, 2004.

Wells, P.S., Anderson, D.R., Rodger, M., et al. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. New England Journal of Medicine 349: 1227-1235, 2003.

Wells, P.S., Anderson, D.R., Rodger, M., et al. Excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to ED by using a simple clinical model and D-dimer. Annals of Internal Medicine 135: 98-107, 2001.

Humphreys, C.W., Moores, L.K., Shorr, A.F., Cost-minimization analysis of two algorithms for diagnosing acute pulmonary embolism. Thrombosis Research 113(5): 275-82, 2004.

ACEP Clinical Policy; Critical Issues in the Evaluation and Management of Adult Patients Presenting with Suspected Lower-extremity Deep Vein Thrombosis. Annals of Emergency Medicine 41: 124-135, 2003.

ACEP Clinical Policy; Critical Issues in the Evaluation and Management of Adult Patients Presenting with Suspected Pulmonary Embolism. Annals of Emergency Medicine 41(2): 257-270, 2003.

D TEST CLINDAMYCIN RESISTANCE *

Effective Date: 
Fri, 03/24/2017
Reviewed: 
Tue, 02/04/2020
Policy: 

Inducible clindamycin resistance in staphylococci can be detected by disk diffusion method using Clindamycin and Erythromycin disks.  D test is performed by disk diffusion, placing a 15-μg  Erythromycin  disk in proximity to a 2-μg Clindamycin disk on an agar plate that has been inoculated with a staphylococcal isolate; the plate is then incubated overnight.

A flattening of the zone of inhibition around the Clindamycin disk proximal to the Erythromycin disk (producing a zone of inhibition shaped like the letter D) is considered a positive result and indicates that the Erythromycin has induced Clindamycin resistance (a positive “D-zone test”). For Erythromycin-resistant isolates, induction tests can help laboratories determine whether results for Clindamycin should be reported as susceptible (when the induction test is negative) or as resistant (when the induction test is positive).

Procedure: 

Materials

Large (15 X 150mm) Mueller Hinton Plate
Small (15 X 100mm) Mueller Hinton Plate
E (erythromycin) 15ug disk
CC (Clindamycin) 2ug disk
Non CO2 incubator
Measuring device (mm)

Quality Control

Run QC with every day of testing.

Positive Control

 Inoculate a small Mueller Hinton plate  (15x100mm) with S. aureus ATCC BAA977, following the Kirby Bauer inoculation method (see Kirby Bauer procedure).
Dispense an E-15 (erythromycin) disk onto the plate. 
Dispense a CC-2 (Clindamycin) disk 15-20 mm from the E-15 disk. 
Incubate overnight at 37 deg C. 
An inhibition zone (See illustration above) should appear around the CC disk. 
Record result in the Manual Testing Log.

Negative Control

Inoculate a small Mueller Hinton plate (15x100mm) with S. aureus ATCC BAA976, following the Kirby Bauer inoculation method (See Kirby Bauer procedure).
Follow steps 2-4 above.
No inhibition zone (see illustration above) should appear around the CC disk.
Record the result in the Manual Testing Log.

Procedure

 On a positive MRSA culture, set up a Kirby Bauer (KB) sensitivity plate following the Wound Culture procedure in the lab manual.
Dispense disks assigned to a wound culture onto the inoculated plate.
Additionally, place an E-15 (erythromycin) disk 15 to 20 mm from the CC-2 (Clindamycin) disk. 
Incubate overnight at 37 deg C.
After incubation, look for an inhibition zone around the CC disk.  See illustration above. If a “D” zone appears, the test is positive for Clindamycin inhibition. Report as ICR positive. If no “D” zone appears and the zone around Clindamycin meets KB sensitivity standards, there is no Clindamycin resistance present. Report as ICR negative.

References

Tankeshwar Acharua:  Inducible Clindamycin Resistance (D Test):  Principle procedure and Interpretation.  Bacteriology August 2, 2013. Microbe Online 10 March 2017.
Woods CR (2009): Macrolide-inducible resistance to clindamycin and the D-test.  Pedriatric Infect Dis J 28:1115-1118

MRSA WOUND CULTURE *

Effective Date: 
Fri, 03/24/2017
Reviewed: 
Tue, 02/04/2020
Revised: 
Wed, 03/29/2017
Policy: 

Purpose and Scope:

The Alere BPB2a SA Culture Colony Test is a qualitative, immunochromatographic assay for the rapid detection of penicillin-binding protein 2a (PBP2a) in isolates identified as Staphylococcus aureus as an aid in identifying methicillin-resistant Staphylococcus aureus (MRSA).  It can identify strains that not only harbor the mecA gene but also produce the protein that confers resistance to methicillin.

Principle of the Method

The Alere PBP2a Culture Colony Test is a rapid immunochromatographic membrane assay that uses highly sensitive recombinant monoclonal antibody fragments (rFabs) to detect the BP2a protein directly form bacterial isolates.  The rFab and a control antibody are immobilized onto a nitrocellulose membrane as two distinct lines and combined with a sample pad, a pink/purple conjugate pad, and an absorption pad to form a test strip.

Isolates are sampled directly from the culture plate and eluted into an assay tube containing Reagent 1.  Reagent 2 is then added and the test strip is placed in the assay tube.  Results are read visually at 5 minutes.

Procedure: 

 

Reagents and Supplies

Materials Provided in the Alere PBP2a SA Culture Colony Test Kit

Test Strips
Reagent 1:  A clear, blue alkaline solution
Reagent 2:  A clear, slightly acidic solution containing sodium azide buffer and surfactants
Assay Tubes
Test Racks

 

Storage and Stability:

Store kit components at room temperature or refrigerated (2-30 deg C). The kit and reagents are stable until the expiration dates marked on their outer packaging.

 

Specimen Required

Specimens are bacterial isolates of Staphylococcus aureus.  The use of fresh (<24 hours) cultures is recommended.  The performance of the test has not been established for use with refrigerated specimens.

Culture Media

S. aureus colonies may be tested from any of the following culture media:

BAP
Columbia Agar
Mueller Hinton Agar

 

Quality Control

Daily Quality Control

PBP2a SA Culture Colony Test has built-in positive and negative procedural controls which are recorded by laboratory personnel for each test run.

 The appearance of a pink/purple line at the control line position is considered an internal positive procedural control.  If capillary flow has occurred, this line will always appear.
In comparison to the color of the control line, the background color on the test strip should be white within 5 minutes.

External Positive and Negative Controls:

External positive and negative controls must be tested and recorded for each new lot and each day of testing.

Control Procedures:

 Subculture the control stains onto a culture plate.:

                      Positive Control:  Staphylococcus aureus :   ATCC #4330

                       Negative Control:  Staphylococcus aureus:  ATCC #25923

 Incubate the plates overnight at 33-35 deg. C for 18-24 hours.
Follow the Procedure below.

 

Procedure

If refrigerated, allow reagents and test strips to equilibrate to room temperature (15-30 deg C) before testing.

The test can be performed from well-isolated colonies on the primary plate if there is sufficient growth, or from a subculture of the isolate.

 Holding the dropper bottle vertically, add two drops of Reagent 1 to an assay tube.
Take one heaped 1 ul bacteriological loop (a heavy inoculum) of sample from well-isolated colonies on the culture plate, place into the tube and thoroughly mix.
Holding the dropper bottle vertically, add two drops of Reagent 2 to the tube.
Vortex briefly.  The blue solution must turn a clear color (if the color does not change, add one more drop of Reagent 2 and mix until the sample turns clear.)
Insert the test strip into the assay tube with arrows pointed downward.
At five (5) minutes, withdraw the test strip from the tube and read the assay result.

For a Negative Sample, a PINK/PURPLE Control Line appears in the top half of the test strip.  No other line appears.

For a Positive Sample, the PINK/PURPLE Contols Line appears AND a second PINK/PURPLE line appears below it in the bottom half of the test strip.  Any Sample Line, even when very faint, is positive.

A test is invalid if the PINK/PURPLE Control line does not appear, whether a Sample Line is present or not.  Repeat invalid tests with a new test strip.  Call Alere Technical Support if the problem persists.

 

 

Key Points: 

References:

 Alere PBP2a Culture Colony Test package insert. Rev. 2 2015/07
Lodise TP, McKinnon PS. Clinical and economic impact of methicillin resistance in patients with Staphylococcus aureus bacteremia. Diagn Microbiol Infect. Dis. 2005 Jun; 52(2):113-22

WOUND CULTURES **

Effective Date: 
Mon, 08/19/2013
Reviewed: 
Tue, 02/04/2020
Revised: 
Thu, 03/23/2017
Policy: 

Purpose and Scope:

Wound infections may be due to a variety of organisms, but are most commonly associated with S. aureus, beta-hemolytic streptococci, P. aeruginosa and enteric Gram negative bacilli. The presence of squamous epithelial cells may indicate that the specimen is superficial and therefore the organisms isolated may not reflect the true etiology of the infection.

Reagents and Supplies:

5% Sheep Blood agar plate
MacCONKEY agar plate
CNA agar plate
Disposable sterile loops
37 C incubator

Procedure: 

Specimen Collection and Transport

Specimens should be collected using a clean, sterile swab and sent in Amies transport medium.

If anaerobic culture is requested, an anaerobic swab placed in an anaerobic transport tube must be collected. If a delay in transport or processing is anticipated, the aerobic swab should be kept at 4 C. If both an aerobic and anaerobic swab are received, both swabs should be kept at room temperature until processed.

Anaerobic swabs, bite wound swabs, ear swabs and eye swabs are to be sent to Quest Diagnostics. Cultures received on Fridays or cultures that require further workup on a Friday are sent to Quest Diagnostics.

Processing of Specimens

 Culture: Media Incubation

Blood Agar (BA)
Colistin Nalidixic Acid Agar (CNA)
MacCONKEY MAC)

Allow plates to come to room temperature before use.

Streak plates for isolation using disposable sterile loop. Incubate 24-48hrs (48hrs if no growth) in 37C incubator.

 Interpretation of Cultures

Examine the aerobic plates after 24 hours incubation. Re-incubate for 24hrs if no growth. Any growth of S. aureus, beta-hemolytic streptococci or Pseudomonas is significant. Enteric gram negative rods and/or enterococci may be significant depending on their number in relation to skin flora and the presence or absence of WBCs on the gram stain.  If Staph aureus is detected, rapid testing for Methicillin Resistant Staph Aureus (MRSA) will be done.

Quantitate the amount of pathogens and any normal skin flora using few, moderate or many estimation. Normal skin flora may include Coagulase negative staph, diptheroids or lactobacillus.

Few = growth only in 1st section
Moderate= growth in 1st and 2nd sections
Many= growth in all sections

Kirby Bauer sensitivities are performed on Staph aureus, enterococci or enteric gram negative rods. If Pseudomonas is detected, sensitivities will be sent to Quest.  If MRSA has been detected, a test for resistance to Clindamycin will be added to the sensitivities.

 

 

 

 

REFERENCES

Textbook of Diagnostic Microbiology, Mahon & Manuselis, 2ndedition, Chapter 27, pages 919-944; Chapter 33, pages 1045-1052; Chapter 35, pages 1083-1113.

Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5thedition, Chapter 3, pages 152-153, 162.

Bailey & Scott’sDiagnostic Microbiology, Forbes, 11thedition, Chapter 59, pages 917-926, and Chapter 63, pages 972-984.

WOUND CULTURES

Effective Date: 
Mon, 08/19/2013
Revised: 
Mon, 02/27/2017
Policy: 

Purpose and Scope:

Wound infections may be due to a variety of organisms, but are most commonly associated with S. aureus, beta-hemolytic streptococci, P. aeruginosa and enteric Gram negative bacilli. The presence of squamous epithelial cells may indicate that the specimen is superficial and therefore the organisms isolated may not reflect the true etiology of the infection.

Reagents and Supplies:

5% Sheep Blood agar plate
EMB agar plate
CNA agar plate
Disposable sterile loops
37 C incubator

 

 

Procedure: 

Specimen Collection and Transport

Specimens should be collected using a clean, sterile swab and sent in Amies transport medium.

If anaerobic culture is requested, an anaerobic swab placed in an anaerobic transport tube must be collected. If a delay in transport or processing is anticipated, the aerobic swab should be kept at 4 C. If both an aerobic and anaerobic swab are received, both swabs should be kept at room temperature until processed.

Anaerobic swabs, bite wound swabs, ear swabs and eye swabs are to be sent to Quest Diagnostics. Cultures received on Fridays or cultures that require further workup on a Friday are sent to Quest Diagnostics.

Processing of Specimens

 Culture: Media Incubation

Blood Agar (BA)
Colistin Nalidixic Acid Agar (CNA)
EMB Agar (EMB)

Allow plates to come to room temperature before use.

Streak plates for isolation using disposable sterile loop. Incubate 24-48hrs (48hrs if no growth) in 37C incubator.

 Interpretation of Cultures

Examine the aerobic plates after 24 hours incubation. Re-incubate for 24hrs if no growth. Any growth of S. aureus, beta-hemolytic streptococci or Pseudomonas is significant. Enteric gram negative rods and/or enterococci may be significant depending on their number in relation to skin flora and the presence or absence of WBCs on the gram stain.

Quantitate the amount of pathogens and any normal skin flora using few, moderate or many estimation. Normal skin flora may include Coagulase negative staph, diptheroids or lactobacillus.

Few = growth only in 1st section
Moderate= growth in 1st and 2nd sections
Many= growth in all sections

Kirby Bauer sensitivities are performed on Staph aureus, enterococci or gram negative rods.

REFERENCES

Textbook of Diagnostic Microbiology, Mahon & Manuselis, 2ndedition, Chapter 27, pages 919-944; Chapter 33, pages 1045-1052; Chapter 35, pages 1083-1113.

Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5thedition, Chapter 3, pages 152-153, 162.

Bailey & Scott’sDiagnostic Microbiology, Forbes, 11thedition, Chapter 59, pages 917-926, and Chapter 63, pages 972-984.

LAB RESULT REQUESTS **

Effective Date: 
Tue, 03/29/2016
Reviewed: 
Wed, 02/05/2020
Policy: 

Students or their representative may request lab results. Lab personnel should instruct the student (or representative) to use the following protocol.

  1. Give the student or representative directions to the medical records office on the 1st floor.
  2. The medical records staff member will confirm the identity of the student or representative and have them fill out an HC 108 Medical Records Release.
  3. Identity of students can be confirmed with the student ID card if it includes the photograph, otherwise a 2nd picture ID will be needed.
  4. Students can authorize release to a representative through use of the HC 108 form. The representative will be authenticated by picture ID if presenting in person.
  5. Parents may access an unemancipated minor's lab results unless the results are related to pregancy, contraception or STD (in which case a release by student would be required).
  6. Authentication of the student's parent is by picture ID and consultation with student's registration record.
  7. Once the HC 108 form is satisfactorily completed  and all identification and/or authentication has been done the student or representative will be provided a copy of the results at that time.

 

 

Syndicate content